Cytochrome c oxidase (COX), or complex IV of the mitochondrial respiratory chain, is a copper- and hemecontaining metalloprotein composed of 13 subunits, 3 encoded by mitochondrial DNA (mtDNA) and 10 by nuclear DNA (nDNA). A number of COX-deficiency disorders are associated with point mutations in mtDNAencoded COX subunits, but very little is known regarding the molecular basis of mendelian-inherited COX deficiency disorders, which display widely varying phenotypes, including both generalized and tissuespecific clinical presentations. In particular, no mutation in any of the 10 nDNA-encoded COX subunits has yet been found. In the last four years, however, mutations have been found in four COX assembly genes - COX10 , SC01, SC02, and SURF1. We believe that other such genes exist, and propose to identify them by screening our large collection of fibroblasts from patients with COX deficiency. We propose to first, screen for "obvious" candidate genes among the known COX structural and assembly genes. Among those cell lines which survive this first screen, we will use functional complementation by a combination of rodent-human monochromosomal hybrids and microcell-mediated chromosomal transfer to identify complementation groups and candidate chromosomal loci. The culprit genes at these loci will be identified by a combination of microsatelle and deletion mapping, coupled with a judicious sequencing of candidate genes in the region. Any new gene thus identified will be characterized as to its role in COX structure, function, and tissue-specific expression. Uncovering the molecular basis of COX deficiency will be useful in terms of pointing the way to diagnosis and treatment of these generally fatal and untreatable disorders. Moreover, these errors will be extremely useful in understanding fundamental biological phenomena, such as COX holoenzyme assembly, COX function, mitochondrial importation, and energy utilization and production. There are no naturally occurring COX mutants in higher eukaryotes other than those causing the diseases outlined above. Thus, elucidating the molecular basis of the COX deficiencies will afford us new and useful insights from both a clinical and scientific standpoint.